The Expression of Lactate Dehydrogenase Is Important for the Cell Cycle of Toxoplasma gondii

In Toxoplasma gondii, lactate dehydrogenase is encoded by two independent and developmentally regulated genes LDH1 and LDH2. These genes and their products have been implicated in the control of a metabolic flux during parasite differentiation. To investigate the significance of LDH1 and LDH2 in this process, we generated stable transgenic parasite lines in which the expression of these two expressed isoforms of lactate dehydrogenase was knocked down in a stage-specific manner. These LDH knockdown parasites exhibited variable growth rates in either the tachyzoite or the bradyzoite stage, as compared with the parental parasites. Their differentiation processes were impaired when the parasites were grown under in vitro conditions. In vivo studies in a murine model system revealed that tachyzoites of these parasite lines were unable to form significant numbers of tissue cysts and to establish a chronic infection. Most importantly, all mice that were initially infected with tachyzoites of either of the four LDH knockdown lines survived a subsequent challenge with tachyzoites of the parental parasites (104), a dose that usually causes 100% mortality, suggesting that live vaccination of mice with the LDH knockdown tachyzoites can confer protection against T. gondii. Thus, we conclude that LDH expression is essential for parasite differentiation. The knockdown of LDH1 and LDH2 expression gave rise to virulence-attenuated parasites that were unable to exhibit a significant brain cyst burden in a murine model of chronic infection. In Toxoplasma gondii, lactate dehydrogenase is encoded by two independent and developmentally regulated genes LDH1 and LDH2. These genes and their products have been implicated in the control of a metabolic flux during parasite differentiation. To investigate the significance of LDH1 and LDH2 in this process, we generated stable transgenic parasite lines in which the expression of these two expressed isoforms of lactate dehydrogenase was knocked down in a stage-specific manner. These LDH knockdown parasites exhibited variable growth rates in either the tachyzoite or the bradyzoite stage, as compared with the parental parasites. Their differentiation processes were impaired when the parasites were grown under in vitro conditions. In vivo studies in a murine model system revealed that tachyzoites of these parasite lines were unable to form significant numbers of tissue cysts and to establish a chronic infection. Most importantly, all mice that were initially infected with tachyzoites of either of the four LDH knockdown lines survived a subsequent challenge with tachyzoites of the parental parasites (104), a dose that usually causes 100% mortality, suggesting that live vaccination of mice with the LDH knockdown tachyzoites can confer protection against T. gondii. Thus, we conclude that LDH expression is essential for parasite differentiation. The knockdown of LDH1 and LDH2 expression gave rise to virulence-attenuated parasites that were unable to exhibit a significant brain cyst burden in a murine model of chronic infection. Toxoplasma gondii is a ubiquitous protozoan parasite that has the ability to infect a variety of warm-blooded vertebrates including humans (1Dubey J.P. J. Am. Vet. Med. Assoc. 1994; 205: 1593-1598PubMed Google Scholar). This parasite has a complex life cycle involving both sexual and asexual multiplication. The sexual cycle occurs exclusively in feline intestinal epithelial cells. In humans and other intermediate hosts, the parasite exists in the following two asexual forms: rapidly dividing tachyzoites and slowly replicating bradyzoites. Initial and acute infection is characterized by the presence of tachyzoites that are normally cleared by the host immune response. In most cases of human toxoplasmosis, infection ultimately becomes chronic when tachyzoites differentiate into bradyzoites. The bradyzoites can remain dormant within tissue cysts protected from the host immune response (2Soete M. Fortier B. Camus D. Dubremetz J.F. Exp. Parasitol. 1993; 76: 259-264Crossref PubMed Scopus (154) Google Scholar). In patients with immunodeficiencies such as AIDS or other malignancies, bradyzoites that differentiate into tachyzoites after release from the cysts can give rise to a recurrent infection that can prove fatal. Congenital malformations are observed when an acute infection is acquired during pregnancy (3Black M.W. Boothroyd J.C. Microbiol. Mol. Biol. Rev. 2000; 64: 607-623Crossref PubMed Scopus (371) Google Scholar). At present, no effective treatment for chronic toxoplasmosis is available. Therefore, cell culture and animal models for the study of chronic Toxoplasma infection are crucial. The differentiation of tachyzoites to bradyzoites can be induced in vitro, although the genetic regulatory signals that control this differentiation are still unknown (2Soete M. Fortier B. Camus D. Dubremetz J.F. Exp. Parasitol. 1993; 76: 259-264Crossref PubMed Scopus (154) Google Scholar, 4Freyre A. J. Parasitol. 1995; 81: 1008-1010Crossref PubMed Scopus (34) Google Scholar). Several studies have identified stage-specific proteins including the cyst wall glycoprotein 1 (5Zhang Y.W. Halonen S.K. Ma Y.F. Wittner M. Weiss L.M. Infect. Immun. 2001; 69: 501-507Crossref PubMed Scopus (90) Google Scholar, 6Cleary M.D. Singh U. Blader I.J. Brewer J.L. Boothroyd J.C. Eukaryot. Cell. 2002; 1: 329-340Crossref PubMed Scopus (154) Google Scholar), surface antigen SAG4A 1The abbreviations used are: SAG4A, surface antigen 4A; DIG, digoxigenin; dsRNA, double-stranded RNA; ENO, enolase; FITC, human RNA; lactate A. M. Boothroyd J.C. Infect. Immun. PubMed Google Scholar), metabolic lactate dehydrogenase PubMed Scopus Google Scholar, J. Parasitol. 2001; Scopus Google Scholar, M. J. Mol. Biol. 2001; PubMed Scopus Google Scholar), and the bradyzoite surface antigen 1 U. J. Mol. Microbiol. 1995; PubMed Scopus Google Scholar). of these genes are developmentally regulated and to be for T. gondii differentiation J.C. M. A. Biol. PubMed Scopus Google Scholar, B. A. Camus D. Mol. Parasitol. PubMed Scopus Google Scholar). dehydrogenase is a that the of to This an when becomes the to under PubMed Scopus Google Scholar). LDH is as a and from of human host PubMed Scopus Google Scholar, L.M. Mol. Parasitol. 2001; PubMed Scopus Google Scholar, A. Mol. Parasitol. PubMed Scopus Google Scholar). the LDH of T. gondii and all of the a into the A. Mol. Parasitol. PubMed Scopus Google Scholar). The of is by an of this is in the from and Toxoplasma L.M. Mol. Parasitol. 2001; PubMed Scopus Google Scholar, A. Mol. Parasitol. PubMed Scopus Google Scholar). In T. gondii, two LDH isoforms are present, LDH1 and LDH2. The genes the isoforms and their products Weiss L.M. Mol. Parasitol. 1995; PubMed Scopus Google Scholar). The genes LDH1 and LDH2 isoforms are developmentally The of LDH2 is in the bradyzoite the of LDH1 is in both bradyzoites and The LDH1 is expressed in that of LDH2 is expressed in bradyzoites. is that LDH1 is by LDH2 during from tachyzoites to bradyzoites as a of of LDH2 PubMed Scopus Google Scholar, Weiss L.M. Mol. Parasitol. 1995; PubMed Scopus Google Scholar). T. gondii and to to and are in is the in bradyzoites L.M. Mol. Parasitol. 2001; PubMed Scopus Google Scholar, J. Microbiol. PubMed Google Scholar). Therefore, which is for be a for for chronic the effective of and of genes has T. gondii to genetic M.W. Boothroyd J.C. J. Biol. PubMed Scopus (34) Google Scholar, Mol. Parasitol. PubMed Scopus Google Scholar). In to these we that the of double-stranded can the expression of T. gondii genes 2002; PubMed Scopus Google Scholar, T. PubMed Scopus Google Scholar). we the in down the expression of the essential genes LDH1 and LDH2 in T. gondii. we have the that the expression of LDH1 and LDH2 is essential for parasite The knockdown of LDH1 and LDH2 expression gave rise to virulence-attenuated parasites that were unable to exhibit a significant brain cyst burden in a murine model of chronic infection. were human that were grown in with and in a of LDH knockdown parasites was the T. gondii, Biol. 1994; PubMed Scopus Google and of were as tachyzoites grown in with To differentiate the parasites into parasites were to for The were with and The was to the M.D. Singh U. Blader I.J. Brewer J.L. Boothroyd J.C. Eukaryot. Cell. 2002; 1: 329-340Crossref PubMed Scopus (154) Google Scholar). were after of of bradyzoite differentiation the A. M. Boothroyd J.C. Infect. Immun. 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To in vivo cyst in the LDH knockdown parasite the mice infected with parasites from and parental as in were for the presence of cysts in the cysts brain were in were in The mice infected with the parental an of cysts brain were when the mice infected with tachyzoites were for the presence of cysts in the cysts were suggesting that the parental the LDH knockdown parasite lines were unable to a significant of cysts in and of T. gondii the expression of of and of expression be observed as the of T. gondii to genetic the expression of essential genes has studies 2002; PubMed Scopus Google Scholar, T. PubMed Scopus Google have that the of is to an and can be used as a genetic for the of In this we that a can be used for LDH1 and LDH2 was genetic M.D. Singh U. Blader I.J. Brewer J.L. Boothroyd J.C. Eukaryot. Cell. 2002; 1: 329-340Crossref PubMed Scopus (154) Google Scholar). The ability to LDH expression has to investigate their significance in parasite initially that the knockdown of LDH expression gave rise to an growth for as by the The of LDH1 expression in tachyzoites and the LDH2 tachyzoite LDH2 is expressed this This that the of dsRNA, the presence of the LDH2 with the cell The which a either LDH1 or LDH2 an effective and the of LDH this was of an of a which be to the other been the knockdown have have to LDH2 expression in the bradyzoite by no were is that the of into bradyzoites by was as as that for transgenic parasite lines were generated and used for the of the and significance of LDH1 and LDH2 in the parasite and parasite lines were generated to the of LDH1 and LDH2 in a stage-specific following the of the expressed The parasite was used for the of LDH1 in bradyzoites. the other was a control parasite for the expression in the of the to the we characterized these parasite in to their parental T. gondii, that these parasite lines expressed the LDH1 and LDH2 dsRNA, as of LDH1 were expressed in tachyzoites and bradyzoites. of LDH2 were in tachyzoites and bradyzoites. the of were to the expression and The was by and tachyzoites exhibited impaired in the The of tachyzoites was that of the parental was that LDH1 by the tachyzoite growth was in PubMed Scopus Google Scholar, L.M. Mol. Parasitol. 2001; PubMed Scopus Google Scholar). Therefore, the is in with these and LDH1 gave rise to and The that the parasites were unable to the mice with a dose that causes 100% in the parental and were unable to a significant of cysts in vivo that the host these parasites and rapidly after an infection parasites. importantly, the infection with protection against a challenge with the parental a dose that 100% of infected is that tachyzoites exhibited growth to the parental parasites and bradyzoites of the parental parasites The of the parasites of the parasite was as compared with the parental parasite parasites were unable to a significant of cysts in vitro and and in vivo that the parasites be in the differentiation The that cysts cysts in the brain were that differentiation is by this parasite was that an LDH that of be to the and the parasite in bradyzoite J. Parasitol. 2001; Scopus Google Scholar, J. Microbiol. PubMed Google Scholar). The of expressed LDH2 and the LDH2 were in as compared with of the parental parasites and Thus, is that of the parasite of LDH2. The of LDH2 expression in bradyzoites be to a of both and is that LDH2 expression have to an and for the bradyzoite and In a murine model in bradyzoite have parasites in the tachyzoites to be cleared by the immune observed for this parasite parasites of are a cysts were we were unable to by a The that infection confer protection against by T. gondii that the infection a to expression of LDH1 in a gave rise to the LDH1 of the of LDH1 expression in the bradyzoite exhibited a to that of the parental parasites. The tachyzoites and bradyzoites a as of the parental parasites. was used as the differentiation was to differentiate in vitro, to that of the parental parasites. is to the cyst wall that in the differentiation (2Soete M. Fortier B. Camus D. Dubremetz J.F. Exp. Parasitol. 1993; 76: 259-264Crossref PubMed Scopus (154) Google Scholar, 4Freyre A. J. Parasitol. 1995; 81: 1008-1010Crossref PubMed Scopus (34) Google Scholar). parasites from the cysts exhibited as compared with the parental parasite suggesting an in cyst In the parasite to a significant of cysts in the of These that the of LDH1 be with the differentiation process, of a we LDH1 in the bradyzoite stage, we have the of LDH1 essential for an with other for the parasite parasite to to This such as of and give into the the and parental bradyzoites. tachyzoites LDH2 in the of the This parasite has a growth as the parental parasite that the presence of to the parasite growth The of the system was effective in the LDH2 as by the of in the of LDH2 in the bradyzoites as compared with other transgenic parasite lines the LDH2 expression in the were that in the parental The that the of LDH2 was expression of LDH2 was that the of LDH2 expression in be to the of an an of the is significant of the This the mice infected with cysts in mice as compared with the mice infected with the parental be that the of cysts in mice infected with was generated by and infection. The by this to be to the of the parasite infection is as as that induced by is to investigate LDH2 in the parasite an an has been that and a as observed for the and Biol. 2002; PubMed Scopus Google Scholar). The of the or and the is the of their in PubMed Scopus Google Scholar). by to subsequent of the an the other the of 2002; PubMed Scopus Google Scholar). and A. D. J. 2002; PubMed Scopus Google Scholar). has been that T. gondii has and T. Microbiol. PubMed Scopus Google Scholar). have the and and are these Thus, is that as as in T. gondii. all LDH knockdown parasites are virulence-attenuated in the animal we the and their products and expressed be In to this we investigate other parasite lines to LDH1 and LDH2 are this is a parasite to growth and differentiation 2002; PubMed Scopus Google Scholar). In we have that the expression of LDH1 and LDH2 is essential for parasite and The of LDH knockdown parasites that are in their to form cysts in vivo the of LDH as a for that tissue cyst The knockdown parasites as a for the of for and for of the for with the for and for The T. gondii host was from the AIDS and of of

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